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. 2021 Sep 30;11:19482. doi: 10.1038/s41598-021-98965-y

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Blocking mutations improve HDR rates. (A) Schematic representation of a desired HDR-generated single base change (orange) 3’ of the PAM. In addition to the desired HDR mutation, a single blocking mutation in the seed region of the guide or PAM to prevent Cas9 re-cleavage was included in donor templates. Each position in the region indicated was changed to every possible alternate base in a unique donor template that also contained the desired HDR mutation. (B) HDR donors for two genomic loci were tested in HEK293 and K562 cells. In each case the donor contained an HDR mutation 3’ of the PAM, with or without a blocking mutation within the region indicated. HDR donors were delivered at 4 µM along with RNP complexes (Alt-R S.p. Cas9 Nuclease complexed with Alt-R CRISPR–Cas9 crRNA and tracrRNA) at 4 µM and with 4 µM Alt-R Cas9 Electroporation Enhancer by nucleofection. Each box represents the rate of perfect HDR including both the desired HDR mutation and blocking mutation (where applicable) as assessed by NGS. Blue indicates a higher HDR frequency, and red indicates a lower HDR frequency. (C) Blocking scores were calculated for 427 samples with known HDR frequencies and used to build a linear model (model = red line, standard error = blue highlight) to determine the optimum HDR efficiency. (D) Schematic representation of four unique HDR mutations that were designed using the Alt-R HDR Design tool either with or without the addition of silent mutations. (E) Four HDR mutations designed using the novel Alt-R HDR Design Tool with ( +) or without (−) silent mutations were tested in HEK293, Hela, and Jurkat cells. RNP complexes (Alt-R S.p. Cas9 Nuclease, Alt-R CRISPR–Cas9 crRNA and tracrRNA) were delivered at 4 µM along with 4 µM Alt-R Electroporation Enhancer and 4 µM donor template in HEK293 and Jurkat cells by nucleofection. RNP complexes (Alt-R S.p. Cas9 Nuclease complexed with Alt-R CRISPR–Cas9 sgRNA) were delivered at 2 µM along with 2 µM Alt-R Cas9 Electroporation Enhancer and 2 µM donor template in Hela cells by nucleofection. Perfect HDR rates were determined by NGS.